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Inflammation in gout: mechanisms and therapeutic targets

Authors:

Alexander So1

Fabio Martinon2

1 Service of Rheumatology, CHUV and University of Lausanne, Lausanne, Switzerland

2 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

Keywords: Gout, Interleukin-1b, monosodium urate crystals, Inflammation

Competing interests:

AS: consultant for Novartis, AstraZeneca, Menarini.

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Key points:

• Inflammatory cytokines, in particular IL-1b are key mediators of gouty inflammation

• Multiple regulatory pathways modulate the activity of the inflammasome and the release of

IL1b

• Diet influences hyperuricemia as well as the inflammatory state of macrophages in gout

• The resolution of gouty inflammation is regulated

Biographies:

Alexander So is head of the Service of Rheumatology at the University Hospital of Lausanne and professor of rheumatology at the University of Lausanne. He obtained his medical training in Cambridge and London, and his PhD at the University of London. His research interest is the role of microcrystals in rheumatic diseases, particularly in gout and in osteoarthritis. His collaboration with Jürg Tschopp led to the use of IL-1 inhibition as a treatment of acute gout and he has participated in a number of clinical and experimental studies of IL1 inhibition in crystal diseases. Fabio Martinon received his Ph.D. from the University of Lausanne, Switzerland, for his work on the characterization of the Inflammasome in the laboratory of Jürg Tschopp. He trained as a postdoctoral fellow in the laboratory of Laurie H. Glimcher at the Harvard School of Public Health, in Boston. He is currently an Associate Professor in the Department of Biochemistry at the University of Lausanne. His laboratory is focused on the characterization of signalling pathways triggered by perturbations of cellular homeostasis and their role in inflammation, inflammatory diseases and cancer.

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Abstract

The acute symptoms of gout are triggered by the inflammatory response to MSU crystals,

mediated principally by macrophages and neutrophils. Innate immune pathways are of key

importance and in particular the activation of the NLRP3-inflammasome leading to release of

IL-1b and other pro-inflammatory cytokines. This review will highlight recent advances in

our understanding of both positive and negative regulatory pathways as well as the genetic

and environmental factors that modulate the inflammatory response. Some of these pathways

can be manipulated and open novel therapeutic opportunities for the treatment of the acute

attack.

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Introduction

Gout has become the most common cause of inflammatory arthritis, and its epidemiology

worldwide points to an increase in incidence and prevalence in both developed and developing

countries 1. It is due to hyperuricemia (serum urate levels > 7mg/L [420 umol/L] leading to

formation and deposition of monosodium urate (MSU) crystals. Clinically, it is characterized by

acute episodes of joint inflammation, usually affecting a single joint, interspersed by symptom free

periods of variable duration. If untreated, it typically progresses to the formation of urate deposits

(tophi) in soft tissues, recurrent attacks of arthritis affecting multiple joints and progressive joint

destruction. Other complications include renal deposits of uric acid that can provoke renal failure

and the formation of renal stones. These and other clinical features have been reviewed recently by

Dalbeth 2.

Gout is now regarded as a prototypical inflammatory disease driven by activation of the innate

immune system and has also been termed an "autoinflammatory disease". However this

classification is misleading, for unlike hereditary autoinflammatory disorders, the acute trigger of

gout is MSU crystals. Uric acid itself is an endogenous and ubiquitous metabolite that is considered

to possess little pro-inflammatory properties, and crystal formation is needed to provoke clinically

observed inflammation.

The study of the underlying mechanisms of gouty inflammation has given us remarkable insights

into the control of the inflammasome and pro-inflammatory cytokine release. Nevertheless, we must

bear in mind some of the other distinguishing features of gout: 1) the attack is usually self-limiting,

and 2) that crystals can be present without an inflammatory response. These observations imply that

there are regulatory mechanisms that modify the acute inflammatory response, and a thorough

understanding of pro- as will as anti-inflammatory pathways may help to develop new strategies for

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treatment. In this review we discuss the recent advances in the field of gout inflammation and new

therapeutic strategies emerging to manage acute gout attacks.

URIC ACID MEDIATED INFLAMMATION

Activation of the NRLP3 Inflammasome by monosodium urate crystals

MSU crystals trigger an inflammatory response from macrophages. The crystals are first taken up

by macrophages and promote the assembly and activation of the NLRP3 inflammasome 4.

Inflammasomes are cytosolic multiprotein complexes that can initiate inflammatory responses 5 6.

Inflammasomes assemble when pattern-recognition receptors such as NLRP3 sense activating

signals that reach the cytosol of the cell. This leads to the oligomerization of the pattern-recognition

receptor and the recruitment to the complex of adaptor proteins and effector enzymes (Figure 1).

NLRP3 inflammasomes are formed by the recruitment of the adaptor ASC and subsequent

recruitment of caspase-1. Following initial oligomerization within the inflammasome, ASC can

further auto-assemble into high molecular weight oligomers. This process, referred to as “prion-

like” polymerization, amplifies the signals and virtually engages all ASC molecules into one active

cellular complex 7. Recruitment and oligomerization of caspase-1 by this structure leads to

activation and proteolytic processing of its substrates.

The cytokines IL-1b and IL-18 are cleaved and activated by caspase-1. In gout, inflammasome

mediated IL-1β-release triggers an important inflammatory response, with vasodilatation and rapid

recruitment of neutrophils to the site of crystal deposition, and thereby drives acute inflammatory

episodes 8.

Additional caspase-1 substrates such as Gasdermins are emerging as downstream effectors of

inflammasome engagement 9. Gasdermins promote cell death upon inflammatory caspases

activation 10, 11. Caspase-1 or caspase-11 cleave Gasdermin D (GSDMD) to release its N-terminal

portion that then polymerizes at the plasma membrane forming cytotoxic pores. These pores alter

cellular integrity and result in cells death by pyroptosis. Pyroptosis, also known as inflammatory

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caspases-mediated cell death, differs from apoptotic caspases-mediated cell death in that it result

into the release of cytosolic content of cells, including a plethora of proinflammatory mediators and

danger signals. Pyroptosis can therefore amplify the inflammatory response and facilitate the

release of cytokines including IL-1b. Whether this pathway contributes to inflammation in gout

remains to be established.

In addition to inflammatory caspases, other proteases can contribute to IL-1 maturation 12. In the

absence of the inflammasome, neutrophils can process proIL-1β by the activity of neutrophil-

derived serine proteases such as proteinase-3 (PR3) elastase and cathepsin G 12, 13. Other serine

proteases can also process IL-1β 14. Some metalloproteinases and granzyme A have also been

proposed to trigger the proteolytic activation IL-1β 15 16. It is unclear whether these pathways

function as an amplifier of the inflammatory reaction, for example in tissues with robust neutrophil

recruitment, or function as backup mechanisms that maintain IL-1β production in condition where

inflammasome proteins are absent or inhibited.

Despite the fact that activation of IL-1β production and NLRP3 inflammasome's role in gout is well

described, the upstream pathway that links monosodium urate crystals to NLRP3 activation are

poorly understood. We can dissect inflammasome engagement into two prerequisite steps: priming

and activation. Relying on two signals is a key feature of most inflammasomes and increases the

specificity of the response and avoids inappropriate firing of the pathway.

MECHANISMS OF INFLAMMATION: PRIMING

Priming, also known as signal 117, controls the expression of all components required for the

assembly and activation of the inflammasome and contribute to the expression of the precursor

proteins that are substrate of inflammatory caspases. This inflammasome-competent stage is

achieved as a result of an inflammatory milieu and can originate by the engagement of innate

immune receptors such as Toll-like receptors (TLRs) or as part of an auto-amplification loop via IL-

1β itself.

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Signalling pathways mediated by cell surface receptors coordinates the innate immune response.

The best known are the TLR family of receptors, and in particular, TLR2 and TLR4 have been

implicated in gouty inflammation. Earlier work showed that TLR2 and TLR4 deficient mice have

an impaired neutrophil response to MSU in the air pouch model 18. A direct interaction between

MSU crystals and the TLRs was postulated, as macrophages deficient for these receptors did not

take up MSU crystals as efficiently as their wild type macrophages. Subsequent data suggest that

TLRs regulate gouty inflammation by recognition of ligands that "prime" monocytes and

macrophages to produce proIL-1β. MRP 8 and 14 are endogenous ligands of TLR4 and are secreted

upon activation of phagocytes. Patients with gout and mice that are injected with MSU produced

high levels of MRP 8 and 14, and genetic deletion of MRP 14 reduced the response to MSU in mice

19. Another ligand that may play a role in macrophage priming in gout is free fatty acids. In a murine

model of gout, arthritis was only observed when mice were injected with both C18 free fatty acids

(FFA) and MSU crystals, and injection of MSU alone or C18 FFA alone was not sufficient to elicit

inflammation. Furthermore, it was demonstrated that TLR2 is the receptor that mediated C18 FFA's

effects on macrophages 20. The mechanisms of how TLRs can regulate inflammation have recently

been reviewed 21.

Other factors such as Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the

complement C5a have been proposed to affect priming in gout. In GM-CSF-neutralized mice, lower

levels of IL-1β were observed following stimulation with MSU crystals. These monocytes also

exhibited decreased expression of NLRP3 and proIL-1β 23. Similarly, treatment with C5a increased

the expression of IL-1β and IL-18 and exacerbated MSU-mediated peritonitis in a mouse model of

gout {An, 2014 #69;Khameneh, 2017 #68}.

While priming is necessary for inflammasome assembly, this step is non-specific and can result

from various conditions and signals that promote an underlining inflammatory response. It provides

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an environment for inflammasome engagement, but it is not sufficient to trigger the inflammasome

pathway per se.

MECHANISMS OF INFLAMMATION: CATALYZING INFLAMMASOME ASSEMBLY

A second signal (signal 2) is required for inflammasome activation. This signal is more specific

than signal 1. It directly drives post-transcriptional and translational aggregation and polymerization

of the inflammasome components. The mechanisms by which monosodium urate crystals trigger

signal 2 to promote NLRP3 activation are still poorly understood. However several steps commonly

found upstream of NLRP3 are involved.

Perturbation of cellular ionic balances, in particular potassium efflux and calcium influx, is the first

feature characteristic of NLRP3 inducers 24, 25. This ionic perturbation is necessary for mitochondrial

reactive oxygen (ROS) generation upstream of NLRP3 inflammasome assembly. ROS production is

also an essential step required for inflammasome formation, it is increased by MSU-mediated

Leukotriene B4 {Amaral, 2012 #22} and may contribute to engage NEK7, a member of the family

of mammalian NIMA-related kinases (NEK proteins). NEK7 directly binds NLRP3 and may be the

common NLRP3 activating ligand 26-28. The important questions that remain to be resolved are how

NEK7 interacts with NLRP3 and the mechanisms by which the crystals promote the ionic changes

that ultimately engage the NLRP3 activating cascade.

IL-1b IS A KEY CYTOKINE IN GOUT

IL-1b is a cytokine that acts on multiple cell types to elicit inflammatory responses 29. It promotes

vasodilatation leading to the recruitment of monocytes and neutrophils to sites of tissue insults, a

response that is crucial in combating infection and restoring tissue homeostasis. However sustained

IL-1b secretion can result in the production of matrix degrading enzymes that breaks down cartilage

and bone30. At the systemic level, IL-1 elicits a fever response by acting directly on the

hypothalamic temperature regulation centre 31.

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IL-1β is mainly produced by innate immune cells and signals to target cells by binding to the IL1

receptor type 1 (IL1R1). Once activated, IL1R1 and its co-receptor IL1 receptor accessory protein

(IL1RAP) recruit a signalling complex that shares components with TLR signalling, leading to the

activation of pro-inflammatory transcription factors including nuclear factor kB (NFkB), as well as

p38, c-Jun N-terminal kinase (JNK) (Figure 2). These transcription factors in turn promote the

transcriptional upregulation of chemokines and proinflammatory mediators that orchestrate the IL-

1-mediated inflammatory response.

It is now widely accepted that IL-1b is a pivotal cytokine in acute gout 32, but a role for IL-1a cannot

be ruled out. IL-1a is released during crystal-induced inflammation and mice with deletion of the

IL-1Β gene are still capable of mounting a neutrophil response 33. The clinical relevance of IL-1 is

supported by data from a number of different clinical studies of IL-1 inhibition 34. Although IL-1

inhibition is not recommended as a first-line anti-inflammatory treatment, the results from clinical

trials (of canakinumab) and cohort studies of patients who have received anakinra as treatment of

acute flares showed a rapid onset of pain relief and it was observed that nearly all treated patients

responded. Patients who had subsequent flares responded equally well when treated with again with

canakinumab 35 and in our personal experience, anakinra was also effective when given for recurrent

flares. The clinical experience with different IL-1 inhibitors in the treatment of gout is detailed in a

later section.

Other cytokines contributing to inflammation in Gout

IL-8, also known as CXCL8 is a macrophage-secreted chemokine that acts principally on

neutrophils. Recent data showed that it is significantly increased during an acute attack in three

different cohorts of patients, and interesting, its levels remained high during the intercritical phase

of gout and also in patients that had concomitant diabetes. In contrast, other co-morbidities that are

commonly seen in gout (such as cardiovascular disease and chronic kidney disease) were not

associated with high IL-8 levels 36. The mechanisms underlying these observations have not yet been

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elucidated, but suggest that neutrophil recruitment and neutrophil activation are key inflammatory

pathways.

Soluble uric acid as a modulator of inflammation

In gout, it is the formation of MSU crystals that trigger acute inflammation, but data show that

hyperuricemia can modulate the inflammatory response. Hyperuricemia by itself, in the absence of

crystals, is able to skew the leukocyte response towards a more inflammatory pattern through

epigenetic modifications of histone methylation. In patients with hyperuricemia, this was shown by

enhanced production of IL-1b and IL-6 and concomitant reduction of IL1RA release 39.

INFLUENCE OF DIETARY FACTORS AND THE MICROBIOME ON GOUT

INFLAMMATION

Clinical observations indicate that dietary factors play a major role in gout, and the link between

patterns of food consumption and hyperuricemia and gout has long been established 40. More

recently, the role of particular foods in triggering an acute attack has been raised, and findings from

an internet-based survey suggest that high purine content increases the risk of an acute attack of

gout five-fold 41. However, the components of food and how they can lead to an attack remains to be

identified. As mentioned earlier, long chain (C18) fatty acids may play a role in priming

macrophages to release IL-1b when they phagocytose MSU crystals 20.

Another mechanism that has been studied recently is the gut microbiome and its interaction with

inflammatory cells and how this affects inflammation. Using a mouse model of gout, it was shown

that germ-free mice showed attenuated MSU-induced inflammation and this effect was reproduced

by antibiotic treatment. They went on to show that these effects were mediated by acetate, a short-

chain fatty acid that is released by gut bacteria. Acetate acts via the macrophage GPR-43 receptor to

modulate inflammasome activation and IL-1b production. Restoring the normal gut flora in germ

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free mice, and the addition of acetate restored the inflammatory properties 42. The same group

further went on to show that a high fibre diet can attenuate arthritis in the same murine model of

gout 43. In that study it was proposed that increased production of acetate and other short chain fatty

acids resulting from the high fibre diet, regulated resolution of inflammation possibly by impacting

neutrophils 43. These studies suggest that environmental factors such as microbial metabolites can

differentially regulate the cell types and pathways of gouty inflammation. The changes in the

microbiome in gout patients have not been extensively studied. There is one report that found that

gout patients had a different bacterial flora compared with control subjects and was similar to that

found in diabetics 44. These findings need to be reproduced in larger cohorts before we can draw

clear conclusions on how the microbiome modulates inflammation in gout.

Although there is great interest in using diet to modulate hyperuricemia, its overall effect is modest.

However, dietary factors may influence gouty inflammation. Recent clinical data suggest that

higher dietary consumption of omega-3 fatty acids is associated with a lower frequency of acute

gout flares 45. Experimentally, �����-3 fatty acids (eicosapentaenoic acid and docosahexaenoic

acid) can inhibit NLRP3 inflammasome activation via a pathway that involves the G-protein

coupled receptors GPR120 and GPR40 and b-arrestin2 46. These findings require confirmation by

intervention trials using omega-3 in gout.

GENETICS OF GOUTY INFLAMMATION

Genetic studies including genome-wide association studies (GWAS) have identified dozen

susceptibility loci associated with hyperuricemia and gout 47. These loci mostly influence uric acid

levels by affecting pathways such as renal and gut excretion of uric acid. While hyperuricemia may

influence oxidative stress and thereby have some impact on inflammatory pathways, whether these

polymorphisms may directly modulate inflammatory responses, beyond promoting uric acid

crystallization, remain to be demonstrated.

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Two studies demonstrated an association of gout with functional variants in CARD8 48, 49. CARD8 is

a potential negative regulator of the NLRP3 inflammasome. It therefore possible that these

polymorphisms may increase inflammasome activity and thereby contribute to the intensity or

duration of NLRP3 engagement in gouty episodes. In addition there is genetic evidence for a role

of TLR4 in gout. Employing a candidate gene approach, two studies, one performed in Han Chinese

and the other in patients with a European ancestry, found a significant association between the same

genetic polymorphism of the TLR4 gene (rs2149356) with gout 50, 51. These polymorphisms may

impact the priming phase of inflammasome engagement or may have a broader impact on the

inflammatory responses in these patients.

One study linked gout incidence with a polymorphism within the gene Peroxisome proliferator-

activated receptor gamma coactivator 1-b (PPARGC1B) {Chang, 2017 #70}. It was shown that this

variant increased NLRP3 and IL-1β expression. Because PPARGC1B function as a regulator of

PPARg , a master regulator of metabolism, this genetic evidence may link metabolic deregulation

with gouty inflammation.

FACTORS CONTRIBUTING TO THE RESOLUTION OF INFLAMMATION

Monocytes and macrophages are the major cellular sources of IL-1, but at the site of inflammation,

neutrophils predominate. The major pro-inflammatory role of the neutrophil and the mechanisms of

interaction between MSU crystals and the neutrophil has been reviewed 52. Interestingly, neutrophils

also probably play a major role in the resolution of acute gout, by the formation of neutrophil

extracellular traps (NET). This process is favoured by high neutrophil concentrations in the

experimental setting (>10 x 106/ ml), and results in the formation of cellular aggregates that contain

cellular debris, DNA as well as neutrophil proteases released into the NETs 53. NET formation is

dependent on the generation of ROS and recent evidence also implicates molecules that regulate

necroptosis via the RIPK3 pathway 54. In the absence of RIPK3, NET formation was inhibited

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completely. Once formed, cellular aggregates containing NETs can degrade a wide range of

inflammatory cytokines rapidly and in experimental models, inhibition of NET formation results in

more severe and persistent gouty inflammation, whereas when NET formation is not impaired, there

is a spontaneous resolution of joint inflammation after day 3 53. These results show that neutrophils

have a dual role in gouty inflammation: in the initial phase when inflammation is amplified by

recruited neutrophils, as well as the resolution of inflammation (Figure 3). Currently, we do not

have any therapies that can modulate this process.

Anti-inflammatory cytokines also contribute to the resolution of the acute inflammatory process. In

animal models, addition of exogenous TGFb1 reduced experimental inflammation 37; in man,

TGFb1, IL-10 and IL1RA were elevated in synovial fluid and are associated with spontaneous

resolution of gouty arthritis {Chen, 2011 #38}. Other factors may contribute to timely resolution of

inflammation in gout. For example, the protein Annexin A1, a potential inhibitor of phospholipase

A2 has been shown to decrease inflammation and promote resolution in mouse models of gout

{Galvao, 2017 #67}.

THERAPEUTIC STRATEGIES TARGETING INFLAMMATION PATHWAYS IN GOUT

Treating gout requires two complementary approaches, one aimed at lowering uric acid level and

the other to reduce inflammation. NSAIDs, colchicine and corticosteroids are commonly used and

are effective in relieving pain and inflammation of the acute attack, however our recent insights into

the biology of inflammation open the way to new therapeutic strategies (Figure 3).

Modulators and inhibitors of the NLRP3 inflammasome

As the NLRP3 inflammasome is a key pathway in the sensing of MSU crystals, strategies that

impede its activation or affect its activity could reduce gouty inflammation. Interestingly, colchicine

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blocks MSU crystals mediated NLRP3 activity in macrophages 32 probably by inhibiting

microtubule-driven rearrangement of mitochondria following NLRP3 engagement with crystals 55.

Other molecules that target key steps leading to NLRP3 assembly have been described to impact

inflammation. The ketone bodies β-hydroxybutyrate suppresses inflammasome activation in

response to monosodium urate crystals 56. These ketone bodies are produced in the liver of mammals

during nutrient deprivation. Hence, starvation attenuated caspase-1 activation and IL-1β secretion in

mouse models caloric restriction 56. Similarly, a ketogenic diet protected rats from urate crystals

mediated gouty flares 57. Mechanistically, β-hydroxybutyrate has been proposed to inhibit potassium

efflux upstream of NLRP3 and to directly impact inflammasome assembly 56. However a possible

effect on priming has also been suggested 57.

Multiple studies in gout have also shown beneficial effects of compounds that inhibit ROS

production and decrease oxidative stress. Epigallocatechin gallate, a potent antioxidant polyphenol

found in green tea has been shown to inhibit neutrophil infiltration and IL-1β secretion in a mouse

model of monosodium urate crystal-mediated peritonitis 58. Morin, a natural flavonol, was found to

impair monosodium urate crystal-induced inflammation in mouse macrophages 59. Rebamipide, a

gastroprotective drug was shown to suppresses monosodium urate crystal-mediated Interleukin-1β

activation and release in human THP-1 cells 60. Further studies are required to interrogate possible

repositioning of this drug in gout. Xanthine oxidase inhibitors used in patients to decrease urate

levels have also been shown to impact directly mitochondrial ROS production thereby inhibiting

urate crystal mediated inflammasome activation 61.

Inhibiting ROS production or potassium efflux are rather non-specific strategies that may have

undesirable effects. Identification of more specific NLRP3 inhibitors could therefore present as

more suitable therapeutics in gout. An example would be MCC950 (also known as CP-456,773 or

CRID3) that has emerged as a potential drug of interest recently. This drug is a diarylsulfonylurea-

containing compound that was initially identified as an inhibitor of extracellular ATP-mediated

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maturation of IL-1β 62. This discovery preceded the initial description of the inflammasome and the

identification of NLRP3 as the main sensor of extracellular ATP signals. More recently it was

demonstrated that MCC950 specifically inhibit the NLRP3 inflammasome 63. It blocks NLRP3-

induced ASC oligomerization in mouse and human macrophages without affecting the activation of

NLRP1, AIM2, or NLRC4 inflammasomes. Several NLRP3 activators, including monosodium

urate crystals where inhibited by MCC950 63, 64, indicating that it may directly act on a conserved

NLRP3 activating mechanism, However, exactly how this drug affects NLRP3 activation is not yet

clear.

Inhibitors of IL-1b maturation

Considerable effort has been taken to develop specific caspase-1 inhibitors. VX-765, an orally

available pro-drug, is the best studied. This drug is rapidly hydrolyzed by plasma and liver esterases

into a potent and selective inhibitor of caspase-1 65. In animals, VX765 ameliorated the severity and

progression of disease in a mouse model of collagen-induced arthritis 66. Caspase-1 inhibitors have

also been shown to decrease IL-1b production and cartilage damage in a model of chronic

destructive joint inflammation 67. However, its effects in gout have not been explored. Of particular

importance will be the specificity of the inhibitor for caspase-1, as caspases share a very conserved

catalytic core and off-target inhibition of apoptotic caspases may cause undesirable consequences.

Based on studies that showed a role for serine proteases in proIL-1b������������ inhibitors

of serine proteases could be of interest, either as monotherapy or in combination with caspase-1

inhibitors in gout. Alpha-1-anti-trypsin (AAT) is a member of the serpin superfamily that inhibits

many serine proteases. Recombinant human AAT-Fc fusion protein was found to be very effective

in a mouse model of gouty arthritis 68. AAT can modulate inflammation at multiple levels and it is

still unclear whether the effects observed in the gout model is directly caused by the inhibition of

IL-1β processing. Yet theses data indicate that AAT-fc and possibly other serine protease inhibitors

could be of therapeutics of interest for gout attacks.

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IL-1 inhibitors

The evidence for the clinical efficacy of IL-1 inhibition in acute gout has been recently reviewed 34.

Two molecules are currently available, but only canakinumab has an indication for acute gout in the

EU. The mechanism of action of the two drugs is different; canakinumab is a specific inhibitor of

IL-1b, and anakinra inhibits both IL-1a and IL-1β binding to the IL1R1 receptor. Their properties

are listed in table 1.

IL-1 inhibition can relieve the acute symptoms of gout in patients who have not responded to

conventional treatments or in whom the use of NSAIDs, colchicine or steroids are contraindicated.

Its use in patients with severe renal and cardiac impairment, a clinical situation that commonly

makes the choice of acute therapy difficult, has not been formally assessed in clinical trials, but in

case series, no severe side effects have been reported 34. In the clinical trials involving canakinumab,

a significant reduction of gout flares was seen for for up to 6 months, however the drug is not

registered for this indication. As drugs have not been tested in large number of patients, their use is

restricted to patients who have "difficult-to treat" disease and their safety in terms of infectious

complications needs to be considered when they are prescribed.

Future considerations

Recent years have shown considerable progress in the understanding of the mechanisms of

inflammation and the role of innate immune sensors in gout, however several question remain

unanswered. Three questions are of particular interest. First the detailed specific mechanism by

which NLRP3 is activated upon exposure to monosodium uric acid crystal is still unclear. Among

the possibilities emerging is that NLRP3 may act as a guardian of cellular integrity that detects

perturbations triggered when innate immune cells attempt at engulfing large particulates 69. This

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would imply that the size and possibly the chemical nature and shape of those crystals may affects

immune responses. Second, the caspase-1 independent mechanisms of IL-1 production, the

proteases engaged and what trigger their activation are still poorly understood. In this context it

would be important to understand at what stage of the response these pathways contribute to the

inflammatory phenotype and what are the cell types orchestrating this inflammasome-independent

response. Finally, a key area of interest involves the mechanisms that initiate the gouty attacks, in

patients that have persistent monosodium urate crystals deposits. It is still unclear whether specific

initiation mechanisms contribute to triggering the inflammatory reaction, possibly by acting on

priming signalling, or whether decrease in negative regulation of NLRP3 engagement are what

promotes the inflammatory cascade. A better understanding of these questions may identify

potential specific therapeutic strategies that will make the prevention and the management of gout

more effective and specific. The study of this old disease provided us with a greater understanding

of inflammatory pathways, solving the remaining questions still bears enormous potential for new

discoveries of pathways and treatments that may impact several inflammatory diseases.

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Figure legends

Figure 1. NLRP3 inflammasome activation by monosodium urate crystals

NLRP3 must be primed before activation. Priming (Signal 1) is mediated by an NF�B–activating

pathways, such as a member of the Toll-like receptors (TLRs). This signaling cascade induces the

expression of functional inflammasome components such as NLRP3. Monosdium urate crystals

provide signal 2 that triggers the assembly of the inflammasome. The interaction of crystals with

the plasma membrane promotes a cellular response that is still poorly understood but includes

hallmarks of NLRP3 activation including potassium efflux through ion channels, and mitochondrial

perturbations leading to the production and release of mitochondrial ROS into the cytosol. Then

NLRP3 activating factors such as NEK7 are engaged promoting NLRP3 oligomerization and

inflammasome assembly. The adaptor ASC is recruited to the inflammasome and nucleates into

prion-like filaments. Caspase-1 is then recruited by ASC and oligomerizes off the ASC filaments

leading to autoproteolytic activation of Caspase-1. Active caspase-1 then promotes the proteolytic

cleavage and maturation of proIL-1b into the biologically active IL-1b. Caspase-1 also promotes

the cleavage of Gasdermin D (GSDMD) to generate an N-terminal cleavage product that

oligiomerizes at the plasma membrane, causing the formation of pyroptotic pores. These pores

disrupt the integrity of the cellular plasma membrane, and may contribute to the release of

inflammatory mediators including IL-1b.

Figure 2. IL-1 signaling links inflammasome activation with the inflammatory cascades

MSU crystals are detected by innate immune cells such as macrophages, monocytes or neutrophils

that respond and produce active IL-1� IL-1β signals through the IL-1R complex, composed of the

IL-1 receptor (IL-1R1) and its co-factor (IL-1RAcP), leading to recruitment of the adaptor MyD88.

The expression of IL1R1 is widespread, present on leucocytes as well as endothelial and synovial

cells. This results in the recruitment of effector proteins such as the IRAKs (mostly IRAK4) and

TRAF6. This triggers the IKK complex and lead to the phosphorylation and degradation of the

23

inhibitor of NFkB, IkB�� Activation of NF�B, turns on the transcription of cytokines and

neutrophil-recruiting chemokines, that will amplify the response and initiate a complex

inflammatory cascade.

Figure 3 Checks and balances of gouty inflammation

Multiple regulatory pathways influence the acute inflammatory response to MSU. The interaction

between macrophage and the neutrophil is important in the regulation of the acute inflammatory

response. Modulators of NLRP3-inflammasome activation include acetate, omega-3 fatty acid and

anti-oxidants can dampen IL-1β release. High concentration of neutrophils will also favor NETosis

and the formation of NET aggregates, which contain proteases capable of degrading inflammatory

cytokines. Finally, the release of TGFb by macrophages also acts as a brake on the inflammatory

response.

Figure 4 Therapeutic targets in gouty inflammation

Pathways leading to IL-1 signaling can be inhibited at many different steps. Example of compounds

and drugs that have been proposed to affect the various steps are shown. While strategies that target

potassium efflux or mitochondrial ROS are quite unspecific, inhibitors of NLRP3 assembly such as

colchicine or inhibitors of IL-1b and IL-1R have already been proven effective in gout.

Table 1: anti-IL-1 drugs

Available IL-1 blockers Drug name Mode of

action Terminal half life

Molecular weight

Administration

anakinra IL-1 receptor antagonist

4-6 hours 17.3 kDa Subcutaneous

canakinumab human anti-IL 1 β monoclonal antibody

26 days 145 kDa Subcutaneous

SIGNAL 1 SIGNAL 2

MSU

K+

K+ K+

K+

K+

K+K+

NEK7

ROS ROS

ROS

ROS

TLRs

plasma membrane

mitochondria

nucleus

pyroptotic pore

ProIL-1β

IL-1β

IL-1β

GDSMD

GDSMD

NFκB In�ammasome components

potassium e�ux

K+

in�ammasome

NLRP3

ASC

Caspase-1

in�ammed joint

uric acid depositions

IL-1RAcP

IL-1R1

IL-1β

MyD88

IRAK1

IRAK4IRAK2

TRAF6

IKKγ

IKKβ IKKα

IκB

NFκB

p65 p50

plasma membrane

nucleus

Innate Immune cells :

Macrophages, Monocytes, Neutrophils

MSU

MSU

MSU IL-1β

IL-1β

IL-1R responding cells :

Endothelial cells, Synoviocytes..

Cytokines

Chemokines

Chemokines

Cytokines

In�ammatory cascades

Neutrophil in�ux

-

Blockers of potassium e�ux

β-hydroxybutyrate

Antioxidants targeting mitochondrial ROS

Epigallocatechin gallate

NLRP3 inhbitors

Colchicine

MCC950

IL-1β processsing inhbitors

VX-765

AAT-Fc

Caspase-1

Serine proteases ProIL-1β

IL-1β inhbitors

CanakiumabIL-1β

Liver product:

Grean Tea molecule:

Plant molecule:

Small molecule:

Orally available drug:

Recombinant protein:

Antibody:

Anakinra

Recombinant protein:

IL-1 receptor inhbitors

IL-1β